Leguminous plant tissue culture was first developed by L. G. Nickell [PNAS (USA), 42, pp. 848-50 1956)]. Sixteen years later, protoplast culture in leguminous plants was developed [O. L. Camborg et al., Physiol. Plant, 30, pp. 125-28 (1974)]. The first successful plantlet regeneration from mesophyll protoplasts was reported in Medicago sativa by [K. N. Kao, Z. Pflanzenphysiol., 96, pp. 135-41 (1980)].
Because of its economic importance, plant research relating to legumes has become the focus of many researchers. Means for introducing new genes into protoplasts and cells of various plants are becoming available [See, for example, J. Paszkowski et al., EMBO Journal, 3, pp. 2717-2722 (1984); M. Fromm et al., PNAS (USA), 82, pp. 5824-28 (1985); J. M. Jaynes et al., Tibtech, pp. 314-20 (1986)]. The transfer of genes can play an important role in improving crops, including legumes by introducing, for example, traits which improve resistance to disease, insects or environmental stress [R.M. Goodman et al., Science, 236, pp. 48-54 (1987)].
The process of plantlet regeneration through protoplast culture is time consuming. And the maintenance of a high differentiation capability of calli after a long period of subculturing is the most important factor to plantlet regeneration from protoplast culture. Researchers have found that in the tissue cultures of Indica rice, certain concentrations of 2,4-D, adding Kinetin can effectively promot the formation of embryo-like structure [D. H. Ling, Acta Botanica Sinica, 29, pp. 1-8 (1987)].
Unfortunately, the procedure of protoplast culture for leguminous plants is not well established and is so time consuming, that the regeneration of plantlets from protoplasts has been achieved in only a small number of legume species: Lotus corniculatus, Onobrvchis viciae, Trifolium repens, Vigna aconitifolia, Trigonella corn, Medicago glutinosa, Coronilla varia [See P. S. Ahuja et al., Plant Cell Rew., 2, pp. 101-04 (1983); P. S. Ahuja et al., Plant Cell Rew., 2, pp. 269-72 (1983); D. Y. Lu et al., Z. Pflanzenphysiol., 107, pp. 59-63 (1982); S. Eapen and R. Gill, Theor. Appl. Genet., 72, pp. 384-87 (1986); A. V. Santos et al., Z. Pflanzenphysiol., 109, pp. 227-34 (1983); A. V. Santos et al., Z. Pflanzenphysiol., 99, pp. 261-70 (1980); D. Y. Lu et al., Chinese Society for Cell Biology, Abstract 105, (1986)]. These represent a total of only 25 species of 11 genera of forage or wild legumen [Q. Zhang, Plant Physiology Communications, 5, pp. 66-76 (1986)].
To date, there have been no reports on the regeneration of cultivated seeded legume plants from protoplasts. With respect to Phaseolus, calli have been obtained from protoplasts only in three species, i.e., P. aureus, P. mungo, P. vulgaris [M. J. Huang et al., Annual Research Reports, (Institute of Genetics, Academic Sinica) 34 (1982); Z. H. Yu et al., Z. Planzenphysiol., 104, pp. 289-98 (1981); L. E. Pelcher et al., Plant Lett, 32, pp. 107-11 (1974)]; but no plantlet formation has ever been successful. In view of the importance of this food crop, a reliable means for regenerating plantlets from protoplasts continues to be needed.